Fuel battery

ABSTRACT

A fuel cell which utilizes the biogenic metabolism to produce a high current density is provided. The fuel cell generates electric power in such a way that the fuel is decomposed stepwise by a plurality of enzymes and those electrons formed by oxidation are transferred to the electrode. The enzymes work such that the enzyme activity of the enzyme involved in decomposition in the early stage is smaller than the sum of the enzyme activities of the enzymes involved in decomposition in the later stage. In the case where a coenzyme is involved, the enzyme activity of the oxidase that oxidizes the coenzyme is greater than the sum of the enzyme activities of the enzymes involved in the formation of the reductant of the coenzyme, out of the enzymes involved in the stepwise decomposition of the fuel.

CROSS REFERENCES TO RELATED APPLICATIONS

The present application claims priority to Japanese Patent Document No.P2002-217802 filed on Jul. 26, 2002, the disclosure of which is hereinincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention generally relates to a fuel cell. Morespecifically, the present invention relates to a fuel cell whichutilizes biogenic metabolism.

The fuel cell basically includes a fuel electrode, an oxidizer electrode(or air electrode), and an electrolyte. The principle of its operationis based on the reverse action of the electrolysis of water. That is,the fuel cell receives hydrogen and oxygen and generates water (H₂O) andelectricity. To be more specific, the fuel electrode is supplied withfuel (hydrogen). Upon oxidation, hydrogen separates into electrons andprotons (H⁺). These protons migrate to the air electrode through the airelectrolyte. At the air electrode, protons react with oxygen suppliedthereto, thereby generating water.

The fuel cell converts fuel's energy directly into electrical energy,thereby functioning as a highly efficient electric power generator. Itcan convert the energy of fossil fuels (such as natural gas, petroleum,and coal) into electric energy very efficiently anytime and anywhere.

Constant efforts have been directed to the research and development offuel cells for large-scale electric power generation. Indeed, the fuelcell mounted on the space shuttle not only generated electric power butalso supplied the crew with water. The fuel cell has proven itself to bea pollution-free electric power generator.

The recent noteworthy development is the fuel cell with a polymericsolid electrolyte which operates at comparatively low temperaturesranging from room temperature to about 90° C. This fuel cell is expectedto find use not only as large-scale electric power generator but also assmall-scale power source for automobiles and as portable power sourcefor personal computers and mobile equipment.

Unfortunately, the above-mentioned fuel cell with a polymeric solidelectrolyte still has problems to be solved despite its advantage ofrunning at comparatively low temperatures. For example, it experiencescatalyst poisoning with CO when it runs with methanol as fuel at aroundroom temperature. It needs a catalyst of expensive noble metal such asplatinum; it suffers energy loss due to crossover; and it encountersdifficulties when it uses hydrogen as fuel.

With the foregoing in mind, there has been proposed an idea of applyingbiogenic metabolism to fuel cells by noting that biogenic metabolismtaking place in an organism is a highly efficient energy conversionmechanism. The term “biogenic metabolism” as used herein embracesrespiration, photosynthesis, and the like. Biogenic metabolism has theadvantage of excelling in power generating efficiency and proceedingunder mild conditions at room temperature.

Respiration is a mechanism consisting of the following steps. First,such nutrients as saccharides, fats, and proteins are incorporated intomicroorganisms and cells. They pass through the glycolytic and TCAcycles involving several enzymatic reactions. (TCA stands fortricarboxylic acid.) During their passage, they give rise to carbondioxide (CO₂) and reduce nicotinamide adenine dinucleotide (NAD⁺) intoreduced nicotinamide adenine dinucleotide (NADH), thereby convertingtheir chemical energy into oxidation reduction energy or electricenergy. The electric energy of NADH is converted directly into electricenergy of proton gradient in the electron transfer system. This step isaccompanied by the reduction of oxygen that forms water. The thusobtained electric energy forms ATP from ADP with the aid of ATPsynthetase. And, this ATP is used for reactions necessary formicroorganisms and cells to grow. Such energy conversion takes place incytosol and mitochondria.

Photosynthesis is a mechanism which consists of steps of taking up lightenergy and reducing nicotinamide adenine dinucleotide phosphate (NADP⁺)into reduced nicotinamide adenine dinucleotide phosphoric acid (NADPH)through the electron transfer system, thereby generating electricenergy. The result is oxidation of water to give oxygen. This electricenergy is used to take up CO₂ for carbon fixation and to synthesizecarbohydrates.

The biogenic metabolism involves the important NADH generating reactionwhich is represented by the formula (3) below.Fuel (reduced form)+NAD⁺

Fuel (oxidized form)+NADH⁺H⁺  (3)(substrate) dehydrogenase (product)

So far, there are known hundreds of dehydrogenases. They play animportant role as a catalyst that performs highly selective conversionof various substrates into products. Their selectivity stems from thefact that the enzyme consists of protein molecules and hence has aunique three-dimensional structure. It follows therefore that fuel takeninto an organism sequentially undergoes reactions involving tens ofdehydrogenases until it is oxidized to CO₂.

The technical idea of applying the biogenic metabolism to fuel cells hasbrought forth the microbial cell which takes out electric energygenerated by microorganisms through an electron mediator and transferselectrons to the electrodes, thereby producing electric current. See,JP-A No. 2000-133297.

Unfortunately, microbes and cells have not only functions to convertchemical energy into electric energy but also other functionsunnecessary for energy conversion. Therefore, the above-mentioned systemcauses electric energy to be consumed for undesirable reactions, therebyreducing the efficiency of energy conversion.

To cope with this situation, there has been proposed a fuel cell basedon an idea of isolating the enzymes and electron mediator involved inreactions from microbes and cells and reconstructing an appropriateenvironment with them in which desired reactions alone take place. Inpractice, however, such a fuel cell merely produces a very low currentdensity on account of the slow reaction rate of enzymes.

The present invention provides a fuel cell which uses the biogenicmetabolism and yet produces a high current density.

SUMMARY OF THE INVENTION

The present invention in an embodiment is directed to a fuel cell whichdecomposes fuel with a plurality of enzymes in stepwise reactions andtransfers electrons produced by oxidation reaction to the electrode,wherein reactions take place such that U(E1)≦U(E2), where U(E1) denotesthe enzyme activity of enzyme-1 to produce decomposition product-1through its decomposition reaction and U(E2) denotes the sum of theenzyme activity of enzyme group-2 to decompose the decompositionproduct-1. The present invention provides a fuel cell which uses thebiogenic metabolism and yet produces a high current density.

The complex enzymatic reactions to decompose fuel stepwise with aplurality of enzymes requires that intermediate products detrimental toenzyme activity should be decomposed immediately. According to anembodiment of the present invention, the enzyme activity of each enzymeis established such that the enzyme activity of a group of enzymes fordecomposition in the subsequent stage is greater than the enzymeactivity of enzyme-1 for decomposition in the preceding stage. Thesystem achieves rapid fuel decomposition.

In the fuel cell defined above, the enzyme-1 is an oxidase in anembodiment.

In the fuel cell defined above, the decomposition reaction by theenzyme-1 is an oxidation reaction which transfers electrons to acoenzyme in an embodiment.

The fuel cell defined above further has a coenzyme oxidase to produce anoxidant of the coenzyme such that U(Co)≧U(E), where U(Co) denotes theenzyme activity of the coenzyme oxidase and U(E) denotes the sum of theenzyme activities of a group of enzymes, out of the plurality ofenzymes, involved in the production of reductant of the coenzyme in anembodiment.

The fuel cell in which electrons are transferred to the coenzyme byoxidation reaction by the enzyme-1 is determined by the enzymaticreaction by the coenzyme oxidase when there is not enough coenzymeoxidase to oxidize rapidly the reductant of the coenzyme. However, thisis not the case for the fuel cell of the present invention in which theenzyme activity U(Co) of the coenzyme oxidase to oxidize the coenzyme isgreater than the sum U(E) of the enzyme activities of a group of enzymesinvolved in oxidation of fuel (or production of reductant of thecoenzyme). In this system, the reductant of the coenzyme is rapidlyoxidized and the resulting electrons are transferred to the electrodethrough the electron mediator according to an embodiment.

A more specific description is given below for a fuel cell which usesmethanol as a fuel. A fuel cell which uses methanol as a fuel includesof a fuel electrode, an air electrode, a proton conducting membraneplaced between the fuel electrode and the air electrode, and an enzymesolution which transfers electrons to the fuel electrode. The enzymesolution contains alcohol dehydrogenase, formaldehyde dehydrogenase,formate dehydrogenase, diaphorase, electron mediator and the like. Thealcohol dehydrogenase, formaldehyde dehydrogenase, formatedehydrogenase, and diaphorase have their enzyme activities which aredenoted respectively by U(ADH), U(FalDH), U(FateDH), and U(DI).

It is assumed that the alcohol dehydrogenase is denoted by enzyme-1. Theenzyme activity U(ADH) corresponding to the enzyme activity U(E1) ofenzyme-1 should be such that U(E1)=U(ADH)≦U(E2)=U(FalDH)+U(FateDH),where U(E2)=U(FalDH)+U(FateDH) is the sum of the enzyme activities ofthe enzymes (formaldehyde dehydrogenase and formate dehydrogenase) whichdecompose the decomposition product (formaldehyde). Then, it is assumedthat the formaldehyde dehydrogenase is denoted by enzyme-1. The enzymeactivity U(FalDH) corresponding to the enzyme activity U(E1) of enzyme-1should be such that U(E1)=U(FalDH)≦U(E2)=U(FateDH), whereU(E2)=U(FateDH) is the sum of the enzyme activities of the enzymes(formate dehydrogenase) which decompose the decomposition product(formic acid). With these factors taken into consideration, the fuelcell which uses methanol as a fuel should preferably satisfy the formula(1) below in an embodiment:0<U(ADH)≦U(FalDH)≦U(FateDH)  (1)

In the above-mentioned fuel cell, the coenzyme oxidase is diaphorase andits enzyme activity U(DI) corresponds to U(Co), and the alcoholdehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase areall involved in production of the reductant of the coenzyme. Therefore,the fuel cell should satisfy the formula (2) below in an embodiment:U(ADH)+U(FalDH)+U(FateDH)≦U(DI)  (2)

In the fuel cell constructed as mentioned above, the alcoholdehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenaseproduce three molecules of NADH in total in the process of catalyzingthe oxidation reaction from methanol (as fuel) to CO₂. In addition, thediaphorase transfers 2 electrons from the thus formed NADH to the fuelelectrode through the electron mediator. The H⁺ which is generated inthese steps reaches the air electrode through the enzyme solution andthe proton conducting membrane. At the air electrode, water is generatedfrom H⁺, oxygen (O₂), and electrons from the external circuit.

The enzyme solution is prepared so that the enzymes produce theirmaximum enzyme activities according to the sequence in which methanol isoxidized (or the enzymes in the order of alcohol dehydrogenase,formaldehyde dehydrogenase, and formate dehydrogenase). Therefore, theenzyme solution permits methanol to be decomposed smoothly and hencepermits NADH to be formed rapidly.

Also, the enzyme solution is prepared such that the enzyme activity ofdiaphorase is greater than the sum of the enzyme activities of alcoholdehydrogenase, formaldehyde dehydrogenase, and formate dehydrogenase.Therefore, the enzyme solution permits electrons to be transferredrapidly from NADH to the fuel electrode without diaphorase becomingsaturated.

The above-mentioned fuel cell of the present invention is characterizedin that electrons are transferred from the coenzyme to the electronmediator. This fuel cell is further characterized in that the electronmediator is vitamin K₃ in an embodiment.

The fuel cell just mentioned above includes a fuel electrode, an airelectrode, a proton conducting membrane held between the fuel electrodeand the air electrode, and an enzyme solution which permits electrons tobe transferred to the fuel electrode, if it is designed to run withmethanol as a fuel. The enzyme solution contains alcohol dehydrogenase,formaldehyde dehydrogenase, formate dehydrogenase, diaphorase, andelectron mediator, and the electron mediator is vitamin K₃.

Vitamin K₃ has an equilibrium redox potential similar to that of thecoenzyme oxidase (e.g., diaphorase) which oxidizes the coenzyme. Itsmoothly transfers electrons to diaphorase and hence it functions as anelectron mediator for fast electron transfer.

As mentioned above, the fuel cell of the present invention involvesvarious kinds of enzymes having activities adjusted such that thereaction to decompose fuel proceeds smoothly and various kinds ofcoenzyme (e.g., NADH) having activities adjusted such that electrons aretransferred to the fuel cell electrode smoothly. In addition, the fuelcell of the present invention employs vitamin K₃ (which functions as anelectron mediator compatible with the dehydrogenase, e.g., DI) so thatelectron transfer takes place rapidly. Thus the fuel cell of the presentinvention brings about rapid reactions and produces a high currentdensity.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic diagram to outline the reaction involved in thefuel cell according to an embodiment of the present invention.

FIG. 2 is a schematic diagram to outline the reaction that takes placein the fuel electrode shown in FIG. 1 according to an embodiment of thepresent invention.

FIG. 3 is a diagram showing the complex enzymatic reaction that takesplace when ethanol is used as fuel according to an embodiment of thepresent invention.

FIG. 4 is a diagram showing the enzymatic reaction that takes place whenglucose is used as fuel according to an embodiment of the presentinvention.

FIG. 5 is a graph which shows how the concentration of NADH changes withtime in Experiment 1 according to an embodiment of the presentinvention.

FIG. 6 is a graph which shows how OCV changes with time in Experiment 2according to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a fuel cell. More specifically, thepresent invention relates to a fuel cell which utilizes biogenicmetabolism. In what follows, the fuel cell of the present invention willbe described in more detail with reference to the accompanying drawingsaccording to an embodiment.

The fuel cell of the present invention is one which utilizes thebiogenic metabolism in an embodiment. As shown in FIG. 1, it includes afuel electrode, an air electrode, a proton conductor to separate thefuel electrode and the air electrode from each other, and an enzymesolution containing enzymes, coenzyme, and electron mediator dissolvedtherein. The enzyme solution is held in a fuel electrode chamber toensure its contact with the fuel cell. The enzyme solution in the fuelcell chamber is continuously supplied with fuel.

In the fuel cell constructed as mentioned above, the enzyme solutioncontains a complex dehydrogenase (including more than one kind ofNAD⁺-dependent dehydrogenase) which oxidizes fuel (say, methanol) intoCO₂ through several steps, while generating NADH from coenzyme NAD⁺. Thethus generated NADH transfers 2 electrons to the fuel electrode throughthe electron mediator with the aid of diaphorase. Current occurs aselectrons reach the air electrode through the external circuit. Theprotons (H⁺) generated by the above-mentioned process migrate to the airelectrode through the enzyme solution with or without proton conductingmembrane. The air electrode generates water from the protons (H⁺), 2electrons supplied from the external circuit, and oxygen.

According to an embodiment of the present invention, a proper measure istaken to optimize the enzymatic activity of the enzyme (such asNAD⁺-dependent dehydrogenase) and/or to select an optimal electronmediator, thereby increasing the rate of migration of electrons to thefuel electrode and also increasing the current density. A detaileddescription is given below, with reference to FIG. 2, of the reaction(shown in FIG. 1) to transfer electrons from the enzyme solution to thefuel electrode.

The enzyme solution contains the following three enzymes dissolvedtherein.

-   -   Alcohol dehydrogenase (ADH for short hereinafter), which        produces formaldehyde and NADH from methanol.    -   Formaldehyde dehydrogenase (FalDH for short hereinafter), which        produces formic acid and NADH from formaldehyde.    -   Formate dehydrogenase (FateDH for short hereinafter), which        produces CO₂ and NADH from formic acid.

The enzyme solution also contains NADH dehydrogenase or diaphorase (DIfor short hereinafter) dissolved therein, which oxidizes and decomposesNADH into NAD⁺ and H⁺. Moreover, the enzyme solution contains anelectron mediator dissolved therein, which receives 2 electrons fromNADH through DI and transfers them to the fuel electrode. Further, theenzyme solution contains the coenzyme NAD⁺dissolved therein, which isnecessary for the NAD⁺-dependent dehydrogenase to participate inreaction in an embodiment.

In the course of fuel decomposition, the enzymes in the enzyme solutionshould exhibit their enzyme activities that meet the followingconditions. It is assumed that enzyme-1 denotes the alcoholdehydrogenase. Then, U(ADH) corresponding to the enzyme activity U(E1)of enzyme-1 should be such that U(E1)=U(ADH)≦U(E2)=U(FalDH)+U(FateDH),where U(E2) represents the sum of the enzyme activities of the enzymes(formaldehyde dehydrogenase and formate dehydrogenase) that decomposethe decomposition product (formaldehyde). Alternatively, it is assumedthat enzyme-1 denotes the formaldehyde dehydrogenase. Then, U(FalDH)corresponding to the enzyme activity U(E1) of enzyme-1 should be suchthat U(E1)=U(FalDH)≦U(E2)=U(FateDH), where U(E2) represents the sum ofthe enzyme activities of the enzymes (formate dehydrogenase) thatdecompose the decomposition product (formic acid). With this taken intoaccount, the fuel cell which consumes methanol as fuel should satisfythe following equation (1) in an embodiment:0<U(ADH)≦U(FalDH)≦U(FateDH)  (1)

According to an embodiment of the present invention, three kinds ofoptimal NAD⁺-dependent dehydrogenases are selected and they are used insuch a way that their enzyme activity sequentially increases as thedecomposition of methanol proceeds. This arrangement permits fastdecomposition of methanol into CO₂ without accumulation of intermediateproducts (such as formaldehyde and formic acid), thereby producing NADHat a sufficiently high rate.

In addition, according to an embodiment of the present invention, it isnecessary that the enzyme activity of DI involved in transfer ofelectrons from NADH to the fuel electrode should be greater than the sumof the enzyme activities of the three kinds of NAD⁺-dependentdehydrogenases involved in production of NADH. The arrangementaccelerates the rate of migration of electrons from the thus producedNADH to the fuel electrode. This is represented by the followingequation (2) in which the enzyme activity of DI is denoted by U(DI)according to an embodiment:U(ADH)+U(FalDH)+U(FateDH)≦U(DI)  (2)

If the enzyme activity of DI is smaller than the sum of the enzymeactivities of NAD⁺-dependent dehydrogenase, the enzymatic reaction of DIrestricts the entire rate of reaction, resulting in slow migration ofelectrons and low current density.

Incidentally, the term U (unit) used herein is an index to represent theenzyme activity which is the rate of reaction for about 1 μmol ofsubstrate per minute at a certain temperature and pH.

The voltage of the fuel cell is established by controlling the redoxpotential of the electron mediator used for each electrode. In otherwords, if a higher voltage is desirable, an electron mediator with amore negative potential should be used for the fuel electrode and anelectron mediator with a more positive potential should be used for theair electrode. Selection of an electron mediator should be made bytaking into account affinity to enzymes, rate of exchange of electronsbetween it and electrode, and stability to inhibiting factors (such aslight and oxygen).

It is desirable from the overall point of view that the electronmediator that acts on the fuel electrode should be vitamin K₃(2-methyl-1,4-naphthoquinone, VK3 for short hereinafter). VK3 has aredox potential of −210 mV (in a solution at pH 7.0), which is slightlymore positive than that of DI (about −380 mV (vs Ag/AgCl) in a solutionat 7.0). DI has flavin mononucleotide (FMN for short hereinafter) as theactive site. This leads to an adequate rate of exchange of electronsbetween DI and electron mediator, which in turn leads to a large currentdensity. When combined with an air electrode, the resulting fuel cellproduces a comparatively large voltage.

If the electron mediator is 1,4-benzoquinone (+93 mV (vs Ag/AgCl) whichhas a more positive potential than VK3, the dispersion of its moleculesinto the solution produces a significant effect, without as much changein current density as expected. Moreover, it reduces the voltage of thefuel cell. Alternatively, if the electron mediator isanthraquinone-2-sulfonate (−549 mV (vs Ag/AgCl) which has a morenegative potential than VK3 and a slightly more positive potential thanDI, the current density decreases but the voltage of the fuel cellincreases. In this case, it is possible to increase the current densityto some extent if the electrode is so constructed as to give a largearea for reaction. Thus, VK3 may be replaced by any electron mediatorhaving an adequate redox potential. Examples of such electron mediatorsthat act on the fuel electrode include compounds having the quinoneskeleton, metal complexes of Os, Ru, Fe, Co, etc., viologen compoundssuch as benzylviologen, compounds having nicotinamide structure,compounds having riboflavin structure, and compounds having nucleotidestructure.

Examples of the electron mediator that acts on the air electrode includeABTS [2,2′-azobis(3-ethylbenzothiazoline-6-sulfonate)] metal complexesof Os, Ru, Fe, Co, and the like.

In this system, electric power is generated in the following manner.First, the enzyme solution is supplied with methanol as fuel. Then, ADHin the enzyme solution catalyzes oxidation of methanol to formformaldehyde. This reaction removes 2 H⁺ and 2 electrons from methanoland forms NADH (reductant of NAD⁺) and H⁺.

Next, FalDH adds H₂O to the formaldehyde and removes 2 H⁺ and 2electrons to form formic acid. This reaction gives rise to NADH and H⁺.

After that, FateDH removes 2 H⁺ and 2 electrons from the formic acid toform CO₂ as the final product. This reaction gives rise to NADH and H⁺.CO₂ as the final product (usually in the form of gas) is removed fromthe enzyme solution. Consequently, CO₂ does not appreciably change thepH of the enzyme solution, and the enzyme solution retains its enzymeactivity.

Finally, DI oxidizes the NADH formed by the above-mentioned process andtransfers electrons to the oxidant of the electron mediator and changesthe oxidant of the electron mediator into the reductant of the electronmediator. In the case where the electron mediator is VK3, the oxidant ofVK3 receives 2 electrons and 2 H⁺ to become the oxidant of VK3. Thereductant of the electron mediator transfers electrons to the fuelelectrode and returns to the oxidant of the electron mediator. The NADHwhich has been oxidized by DI becomes NAD^(+ and H) ⁺, and the resultingNAD⁺is reused while methanol is being decomposed by the NAD⁺-dependentdehydrogenase. The result is that 2 electrons are transferred to thefuel electrode from one molecule of NADH and direct current is produced.

As mentioned above, in the step in which three kinds of NAD⁺-dependentdehydrogenases decompose one molecule of methanol into CO₂, threemolecules in total of NADH are produced. In this stage, H⁺ is extractedfrom H⁺-possessing fuels differing in energy state, and this processconverts the chemical energy into the same substance (NADH). Using thechemical energy possessed by the NADH or by transferring electronspossessed by the NADH to the fuel electrode leads to the fuel cellhaving a high energy conversion rate.

For smooth transfer of electrons from NADH to the fuel electrode, it isdesirable that sufficient electron mediators be present in the enzymesolution for the enzyme activity of DI.

For the above-mentioned enzymes to react steadily and efficiently, theenzyme solution should preferably be kept at approximately pH 7 by meansof tris buffer or phosphate buffer. Also, the enzyme solution shouldpreferably be kept at about 40° C. by a thermostat. The ion strength(I.S. for short hereinafter) should preferably be about 0.3 inconsideration of the electrochemical response. It adversely affects theenzyme activity if it is excessively strong or weak. The above-mentionedpH, temperature, and ion strength will vary with individual enzymes.

The above-mentioned enzymes, coenzymes, and electron mediators may beused in the form of enzyme solution containing them. However, at leastone of them may be immobilized on or near the electrode in the same wayas employed in the field of biosensor. The fuel electrode may beprepared from active carbon or the like (which has a large surface area)in three-dimensionally packed form, so that it has a large area forreaction with the electron mediator. This is effective in increasing thecurrent density further. Moreover, the enzyme may be densely immobilizedon the electrode surface by crosslinking with glutaraldehyde, so thatelectrons are smoothly transferred from the enzyme to the electronmediator near the electrode surface. This helps increase the currentdensity.

Incidentally, the enzymes used in the present invention are notrestricted to those mentioned above and any other enzymes may be used.The above-mentioned ADH, FalDH, FateDH, and DI may be those which aremade comparatively stable to pH and inhibitor by mutation. The enzymefor the air electrode may also be any known one such as laccase orbilirubinoxidase.

The fuel used for the fuel cell of the present invention includes, inaddition to methanol, alcohol (such as ethanol), saccharides (such asglucose), fats, proteins, and organic acids as intermediate productsresulting from glucose metabolism (such as glucose 6-phosphate, fructose6-phosphate, fructose 1,6-bisphosphate, triose phosphate isomerase,1,3-bisphosphoglyceride, 3-phosphoglyceride, 2-phosphoglyceride,phosphoenolpyruvic acid, pyruvic acid, acetyl-CoA, citric acid,cis-aconitic acid, isocitric acid, oxalosuccinic acid, 2-oxoglutaricacid, succinyl-CoA, succinic acid, fumaric acid, L-malic acid, andoxalacetic acid). They may be used alone or in combination with oneanother.

The fuel selected from any of glucose, ethanol, and intermediate productof glucose metabolism mentioned above may be incorporated into a systemin which it is oxidized into CO₂ like methanol if it is combined withone or more adequate enzymes (particularly those involved in the TCAcycle) and the system is operated under optimal conditions. Glucose isparticularly desirable because of its extremely easy handlingproperties.

The enzyme should be selected according to the fuel to be used. Forexample, the enzyme used for the fuel electrode includes glucosedehydrogenase, a series of enzymes involved in the electron transportsystem, ATP synthase, and any enzyme involved in glucose metabolism(such as hexokinase, glucose phosphate isomerase, phosphofructokinase,fructose diphosphate aldolase, triose phosphate isomerase, glycerylaldehyde phosphate dehydrogenase, phosphoglyceromutase, phosphopyruvatehydratase, pyruvate kinase, L-lactate dehydrogenase, D-lactatedehydrogenase, pyruvate dehydrogenase, citrate synthase, aconitase,isocitrate dehydrogenase, 2-oxoglucolate dehydrogenase, succinyl-CoAsynthase, succinate dehydrogenase, fumarase, and malonatedehydrogenase).

FIG. 3 is a diagram showing the complex enzyme reactions that take placewhen ethanol is used as the fuel. In the first stage, ethanol isoxidized into acetaldehyde by the action of alcohol dehydrogenase (ADH).In the second stage, acetaldehyde is oxidized into acetic acid by theaction of aldehyde dehydrogenase (AlDH). In each stage, NAD⁺ (oxidant)is reduced and NADH (reductant) is formed. Electron transfer through theelectron mediator takes place in the same way as methanol as shown inFIG. 2. It is assumed that the enzyme activities of ADH and AlDH arerepresented by U(ADH) and U(AlDH), respectively. Then the equation0<U(ADH)≦U(AlDH) holds for U(ADH) corresponding to U(E1) and U(AlDH)corresponding to U(E2). In addition, the sum of U(ADH) and U(AlDH) issmaller than the enzyme activity U(DI) of DI, as represented by theequation U(ADH)+U(AlDH)≦U(DI).

FIG. 4 is a diagram showing the complex enzyme reactions that take placewhen glucose is used as the fuel. In the first stage for oxidation,β-D-glucose is decomposed into D-glucono-δ-lactone by the action ofglucose dehydrogenase (GDH). Then, D-glucono-δ-lactone is hydrolyzedinto D-gluconate. The D-gluconate is phosphorylated into6-phospho-D-gluconate by hydrolysis of adenosine triphosphate (ATP) intoadenosine diphosphate (ADT) in the presence of glucokinase. In thesecond stage for oxidation, the 6-phospho-D-gluconate is oxidized into2-keto-6-phospho-D-gluconate by the action of phosphogluconatedehydrogenase (PhGDH). In each oxidation reaction, NAD⁺ (oxidant) isreduced and NADH (reductant) is formed. Electron transfer through theelectron mediator takes place in the same way as methanol as shown inFIG. 2. It is assumed that the enzyme activities of GDH and PhDGH arerepresented by U(DGH) and U(PhGDH), respectively. Then the equation0<U(DGH)≦U(PhGDH) holds for U(GDH) corresponding to U(E1) and U(PhDGH)corresponding to U(E2). In addition, the sum of U(GDH) and U(PhGDH) issmaller than the enzyme activity U(DI) of DI, as represented by theequation U(GDH)+U(PhGDH)≦U(DI).

One molecule of ethanol or glucose gives rise to two molecules of NADH;however, it is necessary to increase the energy density of fuel. Toachieve the decomposition of glucose into CO₂, it is necessary to useglucose metabolism. It is necessary that the acetyl-CoA formed byacetaldehyde dehydrogenase (AalDH) should be transferred to the TCAcycle.

A fuel cell that uses glucose as fuel may run with the aid of glucosemetabolism. The complex enzyme reaction based on glucose metabolism isdivided broadly into two categories: glycolysis (decomposition ofglucose and formation of pyruvic acid) and citric acid cycle. They areknown well and hence their description is omitted here.

In the fuel cell in an embodiment of the present invention, the fuelelectrode may be formed from carbon (glassy carbon), Pt, Au, or thelike, and the air electrode may be formed from carbon-filledfluoroplastic which carries a catalyst such as Pt and the like. Inaddition, the air electrode may contain an oxidoreductase such aslaccases, and the like. The proton conducting membrane should preferablybe one such as “Nafion 117” (from DuPont) which is formed fromfluoroplastic and other suitable proton conducting membranes.

As mentioned above, the fuel cell of the present invention in anembodiment is characterized in that the enzyme activity increases as theenzyme reaction proceeds stepwise, as specified by the equations (1) and(2) as above. The enzyme reaction in this way permits decomposition offuel (e.g., methanol) into CO₂ and also permits rapid reactions to formNADH and H⁺ and to decompose NADH. VK3 selected as the electron mediatoraccelerates the rate of migration of electrons from NADH to the fuelelectrode. With the above-mentioned techniques used alone or incombination with one another, the fuel cell based on biogenic metabolismproduces a high current density which has never been achieved. It isdesirable to employ two techniques, so that a higher reaction rate and ahigher current density are attained.

The fuel cell in an embodiment of the present invention utilizes thebiogenic metabolism which is a highly efficient energy conversionsystem. Therefore, it is small in size and light in weight and yet itworks stably at room temperature. Moreover, it permits easy fuelhandling.

The enzymes used for cell reactions may be obtained in the usual way byextraction and purification from cultured cells and microbes whichgenerate the desired enzymes. This helps reduce the production cost ofthe fuel cell.

To further illustrate the invention, and not by way of limitation, thefollowing examples are given.

EXAMPLES

A series of experiments were carried out in the following examples toevaluate the fuel cell of the present invention in an embodiment. In theexperiments, enzymes, coenzymes, electron mediators, and fuels are addedlittle by little (of the order of microliters) so that the change inconcentration and solution temperature is negligible after theiraddition.

Experiment 1

First, the rate at which NADH is formed was investigated to reveal theoptimum enzyme activity of NAD⁺-dependent dehydrogenase.

Example 1

A solution was made from 3 ml of 0.1 M tris buffer (pH 7.0, I.S. =0.3),5 mM of NAD⁺, and 1 M of methanol. The solution was purged with argonwhile stirring. The solution was incorporated with 25 units of ADH, 50units of FalDH, and 75 units of FateDH. After the NAD⁺-dependentdehydrogenase had been added to the solution, change in absorbance withtime was measured by using an ultraviolet-visible spectrophotometer withan optical path length of 1 cm. The wavelength used for measurement was340 nm which is specific for NADH. The concentration of generated NADHwas determined from the change in absorbance. The temperature of theenzyme solution was kept at 40±1° C.

Example 2

The same procedure as in Example 1 was repeated to measure theconcentration of NADH except that the solution of NAD⁺-dependentdehydrogenase was formed from 25 units of ADH, 100 units of FalDH, and200 units of FateDH.

Comparative Example 1

The same procedure as in Example 1 was repeated to measure theconcentration of NADH except that the solution of NAD⁺-dependentdehydrogenase was prepared from 25 units of ADH only, with FalDH andFateDH omitted.

Comparative Example 2

The same procedure as in Example 1 was repeated to measure theconcentration of NADH except that the solution of NAD⁺-dependentdehydrogenase was prepared from 25 units of ADH and 50 units of FalDH,with FateDH omitted.

Comparative Example 3

The same procedure as in Example 1 was repeated to measure theconcentration of NADH except that the solution of NAD⁺-dependentdehydrogenase was prepared from 25 units of ADH only, with FalDH andFateDH omitted, and the solution was incorporated with 50 units of FalDH7.8 hours after the start of measurement and with 75 units of FateDH 9.0hours after the start of measurement.

Comparative Example 4

The same procedure as in Example 1 was repeated to measure theconcentration of NADH except that the solution of NAD⁺-dependentdehydrogenase was prepared from 25 units of ADH, 20 units of FalDH, and15 units of FateDH.

FIG. 5 shows the change in NADH concentration with time which wasmeasured in Examples 1 and 2 and Comparative Examples 1 to 4. Table 1below shows the amount of NAD⁺-dependent dehydrogenase added. The symbol(+) in the column of Comparative Example 3 denotes that the enzyme wasnot added at the start but was added in the course of measurement. TABLE1 Example No. Comparative Example No. 1 2 1 2 3 4 ADH 25 U  25 U 25 U 25U 25 U 25 U FalDH 50 U 100 U — 50 U (+) 50 U 20 U FateDH 75 U 200 U — —(+) 75 U 15 U

It is noted from FIG. 5 that the concentration of NADH steadilyincreases with time in Example 1. By contrast, the rate of NADHformation is low in Comparative Examples 1 and 2 in which theNAD⁺-dependent dehydrogenase is limited to one or two kinds.

The enzyme activity is increased in the sequence of ADH, FalDH, andFateDH, and the ratio of increase is larger in Example 2 than inExample 1. Therefore, the rate of NADH formation is greater in Example 2than in Example 1.

By contrast, the formation of NADH reached the uppermost limit 7 hoursafter the start of measurement in Comparative Example 1 in which thesolution of NAD⁺-dependent dehydrogenase was prepared from ADH only. Therate of NADH formation is higher in Comparative Example 2 than inComparative Example 1; however, the formation of NADH reaches theuppermost limit in Comparative Example 2 in which the solution ofNAD⁺-dependent dehydrogenase was prepared from ADH and FalDH. A probablereason for this is that incomplete decomposition products of methanol(such as formaldehyde and formic acid) accumulate in the enzyme solutionand they alter pH and reduce the enzyme activity. Incidentally, althoughnot shown in FIG. 5, the concentration of NADH did not increaseremarkably 7.8 hours and onward after the start of measurement.

In Comparative Example 3, the rate of NADH formation was low as inComparative Example 1 until FalDH was added but it steeply increasedupon addition of FalDH and FateDH. A probable reason for this is thatformaldehyde remains for a while after the start of measurement but itis decomposed into formic acid and CO₂ upon addition of FalDH andFateDH.

In Comparative Example 4, the enzyme solution contains three kinds ofNAD⁺-dependent dehydrogenase (ADH, FalDH, and FateDH) but their enzymeactivity was sequentially decreased unlike that in Example 1. Therefore,the rate of NADH formation reached the uppermost limit. A probablereason for this is that the enzyme activity of FalDH and FateDH is lowerthan that of ADH and the decomposition of intermediate products(formaldehyde and formic acid) is retarded, which leads to phenomenasimilar to those in Comparative Examples 1 and 2.

The results of Experiment 1 suggest that it is necessary to rapidlydecompose intermediate products which lower the enzyme activity whenmethanol is decomposed into CO₂. To achieve this, it is necessary to usethree kinds of enzymes (ADH, FalDH, and FateDH) as NAD⁺-dependentdehydrogenase in such a way that their enzyme activity sequentiallyincreases in the order mentioned.

Experiment 2

This experiment was designed to find an adequate ratio of enzymeactivities of NAD⁺-dependent dehydrogenase and DI by measuring the opencircuit voltage (OCV) of a three-electrode fuel cell. Thethree-electrode fuel cell has a working electrode of glassy carbon (3 mmin diameter), a counter electrode of Pt wire, and a reference electrodeof Ag/AgCl. It was kept at 40±1° C.

Example 3

The enzyme solution prepared in Example 1 was incorporated with VK3 and200 units of DI, and OCV was measured in the following manner.

A solution was made from 1 ml of tris buffer (pH 7.0, I.S. =0.3), 5 mMof VK3 (oxidant), 5 mM of NAD⁺, and 1 M of methanol. The solution waspurged with argon while stirring. Incidentally, the concentration (5 mM)of VK3 is enough for the enzyme activity of DI. Then, the solution wasincorporated with 25 units of ADH, 50 units of FalDH, and 75 units ofFateDH. The OCV was measured. When the value of OCV became stable, thesolution was incorporated with 200 units of DI. Starting at this point,the OCV was measured continuously.

Example 4

The same procedure as in Example 3 was repeated to measure the OCVexcept that the amount of DI was changed to 400 units.

Comparative Example 5

The same procedure as in Example 3 was repeated to measure the OCVexcept that the NAD⁺-dependent enzyme was 25 units of ADH, with FalDHand FateDH omitted.

Comparative Example 6

The same procedure as in Example 3 was repeated to measure the OCVexcept that the NAD⁺-dependent enzyme was 25 units of ADH and 50 unitsof FalDH, with FateDH omitted.

Comparative Example 7

The same procedure as in Example 3 was repeated to measure the OCVexcept that the NAD⁺-dependent enzyme was 25 units of ADH, with FalDHand FateDH omitted, when OCV measurement started, 50 units of FalDH wasadded when 28.1 minutes elapsed after the start of OCV measurement, and75 units of FateDH was added when 37.3 minutes elapsed after the startof OCV measurement.

Comparative Example 8

The same procedure as in Example 3 was repeated to measure the OCVexcept that the amount of DI was changed to 100 units.

FIG. 6 shows the change in OCV with time which was measured in Examples3 and 4 and Comparative Examples 5 to 8. Table 2 below shows the amount(enzyme activity) of NAD⁺-dependent dehydrogenase and DI used inExamples 3 and 4 and Comparative Examples 5 to 8. The symbol (+) in thecolumn of Comparative Example 7 denotes that the enzyme was not added atthe start of measurement but was added in the course of measurement.TABLE 2 Example No. Comparative Example No. 3 4 5 6 7 8 ADH 25 U 25 U 25U 25 U 25 U 25 U FalDH 50 U 50 U — 50 U (+) 50 U 50 U FateDH 75 U 75 U —— (+) 75 U 75 U Dl 200 U  400 U  200 U  200 U 200 U 100 U 

As shown in FIG. 6, it was observed in all the experiments that theenzymatic reaction proceeded after the start of OCV measurement, therebyforming the reductant of VK3 and reducing the OCV. However, the rate atwhich OCV decreased varies depending on the magnitude of the enzymeactivity of the enzyme added. For example, OCV in Example 3 decreasedmore rapidly than that in Comparative Examples 5 to 8 because DI existedsufficiently relative to NADH formed by NAD⁺-dependent dehydrogenase.The result was different in Example 4 in which the enzyme reaction of DIwas much increased. That is, the transfer of electrons from NADH to VK3accelerated and the OCV decreased rapidly to reach about −0.21 V (vsAg/AgCl) which is the equilibrium redox potential of VK3. This suggeststhat in Examples 3 and 4, there existed sufficient DI for the amount ofNADH produced.

By contrast, the result was different in Comparative Example 8 in whichthe enzyme activity of DI was insufficient for the rate at which NADHwas produced. That is, the OCV decreasing rate was sufficiently high inthe initial stage of measurement but it decreased gradually with time. Aprobable reason for this is that the enzymatic reaction of DI restrictsthe overall reaction rate, thereby increasing the amount of NADH.

The results in Comparative Examples 5 to 7 may be interpreted asfollows. The enzyme activity of DI was sufficient but the enzymeactivity of NAD⁺-dependent dehydrogenase was insufficient, and the NADHforming rate restricted the overall reaction rate, thereby reducing theOCV decreasing rate. In Comparative Example 5, the OCV did not decreaseremarkably 28.1 minutes and onward after the start of measurementalthough this is not shown in FIG. 6.

The results of Experiment 2 mentioned above indicate that the sum ofenzyme activities of NAD⁺-dependent dehydrogenase that forms NADH shouldbe greater than the enzyme activity of DI that oxidizes NADH.

The fuel cell used in Experiment 2 is one which is provided with a fuelelectrode (or working electrode) made of glassy carbon. However, it wasconfirmed that this fuel electrode may be replaced by a Pt electrode, Auelectrode and the like.

The fuel cell used in Experiment 2 is that of three-electrode type.However, it was confirmed that the same result as mentioned above isproduced even when the fuel cell is replaced by one which has a Ptcatalyst as the air electrode and a Nafion membrane as the protonconducting membrane.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

1-14. (canceled).
 15. A fuel cell comprising, the fuel cell capable ofdecomposing fuel with a plurality of enzymes in one or more stepwisereactions and transfers of electrons produced by oxidation reaction toan electrode of the fuel cell, wherein the reactions occur such thatU(E1)≦U(E2), where U(E1) denotes the enzyme activity of enzyme-1 toproduce decomposition product-1 through its decomposition reaction andU(E2) denotes the sum of the enzyme activity of enzyme group-2 todecompose the decomposition product-1.
 16. The fuel cell as defined inclaim 15, wherein the enzyme-1 includes an oxidase.
 17. The fuel cell asdefined in claim 15, wherein the decomposition reaction by the enzyme-1includes an oxidation reaction which transfers electrons to a coenzyme.18. The fuel cell as defined in claim 17, which further includes acoenzyme oxidase to produce an oxidant of the coenzyme such thatU(Co)≧U(E), where U(Co) denotes the enzyme activity of the coenzymeoxidase and U(E) denotes the sum of the enzyme activity of a group ofenzymes associated with the plurality of enzymes that are involved in aproduction of reductant of the coenzyme.
 19. The fuel cell as defined inclaim 17, wherein the coenzyme produces an oxidant that includes NAD⁺and a reductant that includes NADH.
 20. The fuel cell as defined inclaim 18, wherein the oxidant of the coenzyme is formed by a coenzymeoxidase that includes diaphorase.
 21. The fuel cell as defined in claim17, wherein the coenzyme transfers electrons further to an electronmediator.
 22. The fuel cell as defined in claim 21, wherein the electronmediator includes vitamin K₃.
 23. The fuel cell as defined in claim 16,wherein the fuel is at least one species selected from the groupconsisting of alcohols, saccharides, fats, proteins, and organic acids.24. The fuel cell as defined in claim 22, wherein the fuel is at leastone species selected from the group consisting of methanol, ethanol, andglucose.
 25. The fuel cell as defined in claim 16, wherein the fuelincludes methanol and the enzymes that decompose the fuel stepwise areselected from the group consisting of alcohol dehydrogenase,formaldehyde dehydrogenase, formate dehydrogenase and mixtures thereof.26. The fuel cell as defined in claim 16, wherein the fuel includesethanol and the enzymes that decompose the fuel stepwise are alcoholdehydrogenase and aldehyde dehydrogenase.
 27. The fuel cell as definedin claim 16, wherein the fuel is glucose and the enzymes that decomposethe fuel stepwise are selected from the group consisting of glucosedehydrogenase, gluconokinase, phosphogluconate dehydrogenase andmixtures thereof.
 28. The fuel cell as defined in claim 18, wherein thefuel includes methanol, the enzymes that decompose the fuel stepwise areselected from the group consisting of alcohol dehydrogenase,formaldehyde dehydrogenase, formate dehydrogenase and combinationsthereof, the coenzyme oxidase that oxidizes the coenzyme includesdiaphorase, and the conditions specified by equations (1) and (2) beloware satisfied and defined as follows:0<U(ADH)≦U(FalDH)≦U(FateDH)  (1)U(ADH)+U(FalDH)+U(FateDH)≦U(DI)  (2) where U(ADH) denotes an enzymeactivity of the alcohol dehydrogenase, U(FalDH) denotes an enzymeactivity of the formaldehyde dehydrogenase, U(FateDH) denotes an enzymeactivity of the formate dehydrogenase, and U(DI) denotes an enzymeactivity of the diaphorase.